The detection of nucleic acids in samples, in particular biological samples, is well known in the fields of research, diagnosis (in particular of disease and genetic conditions), forensics and detection of microorganisms (for example for hygiene, environmental monitoring or threat reduction, where potentially harmful microorganisms such as bacteria are required to be detected rapidly).
Lateral flow devices (LFDs) have long been used in the field of diagnostics to detect target analytes such as proteins including hormones, antigens, antibodies etc. In these devices, a liquid sample containing or suspected of containing the analyte flows along a membrane, where it encounters labels, labelled binding partners and/or immobilised binding partners, in a sequence whereby a detectable visible signal is developed on the membrane depending on the presence or absence of the analyte in the sample.
The volume of liquid required to cause a sample to effectively flow along an LFD is generally quite significant. The membrane used as a substrate for the LFD is porous and will generally absorb significant amounts of liquid. Furthermore, the liquid flow must be sufficient to ensure that the labelled moieties are carried through to the detection zone on the device.
LFDs may also be used to detect analytes that comprise nucleic acids such as RNA or DNA. In this case, the binding partners for the analytes will include oligonucleotides that hybridise to the specific target sequence or, alternatively, binding partners for binding agents that have been incorporated into the RNA or DNA, for instance during a preliminary amplification reaction. For instance, nucleic acid amplification reactions may also be used to incorporate a binding agent, such as biotin, into the target so as to facilitate capture in the detection zone. Where biotin has been incorporated into a target nucleic acid, the presence of streptavidin or anti-biotin antibodies in the detection zone on the LFD will result in capture of biotin-labelled target nucleic acids in the capture zone.
Labelling may be effected using either labelled probes that also hybridise, for instance, to the target sequence so as to produce a visible signal when the target becomes immobilised in the detection zone. Labelling may also be achieved by incorporating a label into the target sequence, for instance during an amplification reaction, where labelled primers are used to generate an intrinsically labelled product. Suitable labels are well known in the art; for example, there are chemical or biochemical labels such as fluorescent labels, which include, for instance, fluorescein or fluorescein derivatives, or cyanine dyes, or labels that may be detected enzymatically such as digoxigenin. Alternatively or additionally, labels may comprise particulate labels such as gold, silver, and latex beads or particles, which produce a visible signal directly. These may be arranged to interact with target nucleic acid in the detection zone. In order to achieve this, the particles themselves will be labelled, for example conjugated to, moieties that interact with the target nucleic acid (for example other nucleic acids that hybridise to the target nucleic acid), or they may be conjugated to a binding agent such as streptavidin, that interacts with a binding partner such as biotin, which has been incorporated into the target nucleic acid sequence.
In fact, in most cases, the concentration of target nucleic acid in a biological sample is low, certainly below that at which a visible signal may be generated directly on a LFD. Thus, as a preliminary step, amplification of the nucleic acid is generally required.
Nucleic acid amplification techniques are a powerful tool in this area. There are many techniques, some of which are carried out isothermally and some of which require thermal cycling such as the polymerase chain reaction, which allow very small amounts of target nucleic acid in a sample to be amplified to detectable levels.
However, the extreme sensitivity of these techniques means that they are very prone to contamination or cross-contamination. Even a very small amount of contaminating nucleic acid may be subject to amplification in these methods, leading to false positives.
Many attempts have been made to address this problem, focussing principally on ensuring that the sample is treated in an environment isolated from the amplification process, as far as possible. Thus, methods for carrying out an amplification reaction and detecting the amplification product in a homogenous reaction, where the reaction vessel does not have to be opened, have been developed.
For example, WO2004/065010 relates to a microfluidic system for isolation and amplification of DNA and detection of DNA on a lateral flow detection strip. DNA from lysed bacterial cells is captured on a solid substrate through which amplification reagents are pumped. Amplified DNA is then pumped over a lateral flow strip. The system requires relatively complex apparatus, since pumping of reagents is required. The user is required to pipette the various solutions needed during the process onto the card, which carries significant risks of contamination.
US2011/0039261 also relates to a test system for nucleic acid analysis, with amplification of DNA and detection of amplification products on a lateral flow test strip. Again, it is necessary for the sample to be transferred between different cavities in the device, by some pumping mechanism. Running buffer to facilitate the detection on the lateral flow device is added to the system after the amplification sample has been passed to the test strip.
There is a need for an integrated system that allows for analysis to be carried out rapidly using simple apparatus, such that a relatively unskilled user can operate the system without the need for onerous manual operations, with minimal contamination risk and with maximal efficiency.
The applicants have developed a device that allows chemical and biochemical reactions such as nucleic acid analysis to be carried out in an isolated unit, which may be disposable, with minimum contamination risk. Aspects of a similar device were described in WO2011/051735. Significant improvements have now been provided, with advantages as described herein.